DNA microarrays are a primary tool for DNA genotyping because of their capacity to detect multiple targets simultaneously. However, DNA microarrays require fluorescent labeling and long incubation steps which limit their applicability. Furthermore, DNA microarrays are not capable of detecting DNA targets in the presence of excess background DNA. This capacity is essential in multiple biomedical applications, such as tumor genotyping, microbiology testing and non-invasive prenatal genetic analysis. Here, we propose development of a novel microarray based on Twist-Biosensor (TBS) technology that will be label-free and more than 20 times faster than standard microarrays. Additionally, the new technique will be capable of detecting DNA in the presence of 20-fold excess background DNA, making it ideal for difficult genotyping situations. In this application, we propose the development of novel microarrays to dramatically improve and simplify tumor genotyping for research and clinical applications. Available techniques used to detect somatic mutations are either limited in their multiplexing capacity, such as real-time PCR and mass spectroscopy, or time consuming and labor intensive such as Next-Gen, Sanger and Pyro-sequencing. Twist-Biosensor is a novel microarray technique in which hybridization is detected with single molecule resolution. In addition, Twist- Biosensor applies disrupting torsional stress to DNA hybrids, a novel strategy that gives the microarrays extremely high sequence selectivity. Using these unique properties, we will develop highly multiplexed microarrays to detect somatic mutations that will be accurate, rapid, and cost- effective. The overall aim of this proposal is to demonstrate the capabilities of Twist-Biosensor microarrays for the detection of multiple mutations in the presence of excess background DNA. We will develop prototype devices to detect mutations of the BRAF and KRAS genes and test them using tumor samples. Aim 1 focuses on demonstrating rapid (3 hours) and accurate detection of a single mutation in samples containing 20-fold excess background DNA. Aim 2 focuses on improving TBS detection by integrating the biosensors in a digital sensor array. The new system will have high multiplexing capability, will use inexpensive equipment and will be easy to automate. Our ultimate goal is to produce a flexible platform for biomedical research and clinical applications.